反応 #516920

反応方程式

GDP-Fuc

Galβ1,3GlcNAc

NaF

NADPH

GDP-mannose

→

Galβ1,3(Fucα1,4)GlcNAc

溶媒

2-propanol

反応条件

後処理

1. 1
   workup.ADDITIONdiluted to 3 mL
2. 2
   workup.ADDITIONα1,3/4-Fucosyltransferase (0.01 U) was added to the mixture
3. 3
   ろ過The mixture was filtered
4. 4
   その他The fractions were collected
5. 5
   その他The residual material was purified with a column of Sephadex

実験手順

A mixture of GTP Na salt (6.0 mg, 10 μmol), Man-1-P K salt (3.5 mg, 10 μmol), Galβ1,3GlcNAc (3.8 mg, 10 μmol), NaF (0.42 mg, 10 μmol), NADPH (9.4 mg, 10 μmol), PEP K salt (4.1 mg, 20 μmol), MgCl2—6H2O (2.6 mg, 10 μmol), MnCl2—4H2O (2 mg, 10 μmol), 2-propanol, (50 μL), ADH (12 U), PK (200 U), PPase (100 U), crude enzyme preparation of GDP-mannose pyrophosphorylase (1.0 mL), and crude enzyme preparation of GDP-Fuc producing enzyme (1.0 mL) in 100 mM tris buffer (pH 7.5) and diluted to 3 mL. α1,3/4-Fucosyltransferase (0.01 U) was added to the mixture and the resulting mixture was stirred under Ar for three days at room temperature. The mixture was filtered and the filtrate was applied to a column of Dowex 1-X8 [OH−) form followed with a column of Dowex 50W-X8 [H+] with water. The fractions were collected and lyophilized. The residual material was purified with a column of Sephadex G-25 (superfine) with water. The appropriate fractions were pooled and lyophilized to give Galβ1,3(Fucα1,4)GlcNAc. Its 1H-NMR spectrum was in good agreement with that reported. [Dumas et al., Biomed. Chem. Lett., 1:425 (1991).]

出典